PROJECT SUMMARY/ABSTRACT Chlamydia is the most commonly reported sexually transmitted infection in the US. Infections are usually asymp- tomatic and often go untreated which can lead to pelvic inflammatory disease, ectopic pregnancy, and infertility. Chlamydia trachomatis is an obligate intracellular pathogen that establishes its replication niche in host epithelial cells within a membrane-bound compartment called the inclusion. C. trachomatis intracellular replication de- pends on the secretion of bacterial effector proteins through a type III secretion system. These effectors are translocated into the cytosol of the host cell and some effectors, called Inc proteins, are embedded in the inclu- sion membrane. Inc proteins have tails facing the host cell cytosol and can interact with and manipulate host cell molecules. Our lab has shown that the inclusion membrane establishes close contacts with the endoplasmic reticulum (ER), which we termed ER-inclusion membrane contact sites (MCS). In uninfected cells, the ER es- tablishes MCS with various organelles allowing for the non-vesicular transfer of lipids, ion exchange, and cell signaling. Previously, we have shown that ER-inclusion MCS harbor the Chlamydia Inc protein IncD, the host ceramide transfer protein CERT, and the ER-resident VAP proteins and proposed a role for the IncD-CERT-VAP complex in the transfer of ceramide to the inclusion. Recently, we have focused our efforts on understanding the mechanisms by which the ER and the inclusion membranes are brought and maintained in close proximity. In a recent publication, I have shown that the Inc protein IncV interacts with VAP by molecular mimicry of eukaryotic FFAT motifs. My data indicate that while the IncV-VAP complex is sufficient to mediate ER-inclusion MCS for- mation, it is not essential, suggesting that redundant mechanisms exist. Here, I propose to further investigate the mechanisms used by Chlamydia to tether the ER to the inclusion. Specifically, I will determine the mechanism supporting the IncV-dependent formation of MCS (Aim1) and characterize the respective contributions of the IncV-VAP and IncD-CERT-VAP complexes (Aim2). My central hypothesis is that ER-inclusion MCS formation relies on the recruitment of the ER-resident VAP protein through interaction with the Chlamydia Inc proteins IncV and IncD. Since ER-inclusion MCS are necessary for the Chlamydia lifecycle, my approach may reveal novel therapeutic targets for therapeutic intervention.